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Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.

Assuntos

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BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Assuntos

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Stress is a widespread problem in today's societies, having important consequences on brain function. Among the plethora of mechanisms involved in the stress response at the molecular level, the role of microRNAs (miRNAs) is beginning to be recognized. The control of gene expression by these noncoding RNAs makes them essential regulators of neuronal and synaptic physiology, and alterations in their levels have been associated with pathological conditions and mental disorders. In particular, the excitatory (i.e., glutamate-mediated) neurotransmission is importantly affected by stress. Here, we found that loss of miR-26a-5p (miR-26a henceforth) function in primary hippocampal neurons increased the frequency and amplitude of miniature excitatory currents, as well as the expression levels of the excitatory postsynaptic scaffolding protein PSD95. Incubation of primary hippocampal neurons with corticosterone downregulated miR-26a, an effect that mirrored our in vivo results, as miR-26a was downregulated in the hippocampus as well as in blood serum-derived small extracellular vesicles (sEVs) of rats exposed to two different stress paradigms by movement restriction (i.e., stress by restraint in cages or by complete immobilization in bags). Overall, these results suggest that miR-26a may be involved in the generalized stress response and that a stress-induced downregulation of miR-26a could have long-term effects on glutamate neurotransmission.

Assuntos

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In the last few decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP-tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) that are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e., GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation, but also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.

Assuntos

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Astrocytes use gliotransmitters to modulate neuronal function and plasticity. However, the role of small extracellular vesicles, called exosomes, in astrocyte-to-neuron signaling is mostly unknown. Exosomes originate in multivesicular bodies of parent cells and are secreted by fusion of the multivesicular body limiting membrane with the plasma membrane. Their molecular cargo, consisting of RNA species, proteins, and lipids, is in part cell type and cell state specific. Among the RNA species transported by exosomes, microRNAs (miRNAs) are able to modify gene expression in recipient cells. Several miRNAs present in astrocytes are regulated under pathological conditions, and this may have far-reaching consequences if they are loaded in exosomes. We propose that astrocyte-derived miRNA-loaded exosomes, such as miR-26a, are dysregulated in several central nervous system diseases; thus potentially controlling neuronal morphology and synaptic transmission through validated and predicted targets. Unraveling the contribution of this new signaling mechanism to the maintenance and plasticity of neuronal networks will impact our understanding on the physiology and pathophysiology of the central nervous system.

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CDNF is a recently described evolutionary conserved neurotrophic factor reported to be of relevance for the treatment of Parkinson's disease. Treatment with recombinant CDNF showed neurorestorative and neuroprotective effects on dopaminergic neurons in Parkinsonian animal models. Similar results are obtained using adeno-associated viral (AAV) vectors for CDNF expression in these animal models; however, the extent of the transduced brain tissue is difficult to assess due to the lack of reporter genes in the vectors used. Here, we describe two bicistronic lentiviral plasmids based on the Δ1D/2A and IRES elements for the expression of EGFP and rat CDNF, in order to track the transduced cells expressing CDNF with EGFP fluorescence. Transfected heterologous cells or transduced neurons with these vectors are easily identified by EGFP fluorescence and CDNF expression results in its recruitment to the endoplasmic reticulum (ER) by both bicistronic vectors. CDNF immunostaining is also observed in the Golgi apparatus when expressed in heterologous cells or hippocampal neuronal cultures; however, colocalization with a dense core secretory vesicle marker was scarce. Additionally, we showed that the expression of CDNF inhibited dendrite formation in hypothalamic neurons, suggesting that CDNF expressed by these bicistronic lentiviral vectors is functional and could have a role in neuronal morphology. The bicistronic lentiviral plasmids developed here could be of use to study the effect of rat CDNF at the cellular level or to better delineate the perikarya of neurons transduced with lentiviral vectors in animal models of Parkinson's disease.

Assuntos